Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Small molecules can act very fast, can be very specific, and can help to distinguish the temporal order of molecular steps and the hierarchical regulation of biological processes. Because small molecules can alter the function of a specific gene product, they can be used in a manner analogous to the use of inducible dominant or homozygous recessive genetic mutations. A large body of biochemical literature is based on the past use of small molecule antagonists that were employed in "reverse chemical genetics" approaches to conditionally eliminate protein function, and on that basis to subsequently identify the target, its mechanism of action, and its regulation. Thus, ouabain helped to define the catalytic cycle of the NaK-ATPase, cytochalasin B was instrumental in defining the molecular basis for insulin's action to stimulate glucose uptake, and analogs of amiloride were used to purify and define the epithelial Na channel. There is a need to develop "forward chemical genetics" in order to discover small molecules that partner with key elements in a pathway of interest. This proposal is supported by preliminary data that establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Iron deficiency remains the most prevalent nutritional problem in our country, yet recent identification of the gene responsible for hereditary hemochromatosis indicates that 1 in 20 Caucasians carry the defective allele and thus 1 in 400 may be susceptible to iron overload. Increased knowledge about the transport factors and how they protect against iron deficiency and overload is essential to more broadly address these significant health problems. Using the cell-based fluorescence assay, we propose to: 1) Perform chemical genetic screens for selective inhibitors of different pathways of iron transport using combinatorial libraries; 2) Characterize the compounds identified to block iron uptake with highest potency; 3) Develop structure-activity profiles on compounds of interest and identify their targets. The goals of this project are to discover small molecule inhibitors of iron transport using chemical genetics and to use these reagents to advance our understanding of the factors, mechanisms, and regulation of different pathways of iron uptake.